o:38969 Mehanizmi interakcija mezenhimalnih matičnih ćelija i makrofaga u regulaciji stres eritrocitopoeze doktorska disertacija Mechanisms of interaction between mesenchymal stem cells and macrophages in the regulation of stress erythropoiesis : doctoral dissertation sr smanjene oksigenacije tkiva što za posledicu ima aktivaciju različitih mehanizama u ciljuodržavanja adekvatnog snabdevanja tkiva kiseonikom. Povećane potrebe organizma za eritrocitimau navedenim uslovima dovode do aktivacije stres eritrocitopoeze (SE). Tokom procesa SE dolazi domigracije eritroidnih progenitora iz koštane srži u slezinu gde pod uticajem signalnog molekulakoštanog morfogenetskog proteina (engl. bone morphogenetic protein 4, BMP4) ove ćelijepoprimaju karakteristike specifičnih stres eritroidnih progenitora i dolazi do njihove nagleekspanzije. U koštanoj srži i slezini eritrocitopoeza se odvija unutar specijalizovanih eritroblastnihostrva u kojima centralno mesto zauzimaju makrofagi koji imaju ključnu ulogu u formiranju zreliheritrocita. U uslovima povećanih potreba za eritrocitima monociti se pod uticajem hemokina,izlučenih iz crvene pulpe slezine, iz koštane srži regrutuju u slezinu gde se diferenciraju umakrofage eritroblastne niše. Pored makrofaga, značajnu komponentu hematopoezne mikrosredinečine mezenhimalne matične ćelije (engl. mesenhimal stem cells, MSC) koje produkuju brojnecitokine uključene u imunomodulaciju, proliferaciju, diferencijaciju i migraciju hematopoeznihćelija. U zavisnosti od mikrosredine MSC i makrofagi ostvaruju interakcije posredstvom različitihsignalnih molekula. Međutim, uloga makrofaga i mehanizmi njihovih interakcija sa MSC u okvirutkivno-specifične mikrosredine tokom SE do sada nisu razjašnjeni. Cilj ove studije bio je da seispita uloga makrofaga i njihovih tkivno-specifičnih interakcija sa MSC u regulaciji procesa SE, sposebnim osvrtom na učešće azot-monoksida (engl. nitric oxide, NO), vanćelijskih nukleotida ipurinergičkog signalnog sistema u ispitivanim interakcijama. U tu svrhu korišćeni su miševi,mužjaci Balb/c soja, kao i mono i ko-kulture RAW264.7 ćelija i MSC izolovanih iz koštane srži islezine miša. Miševi su bili podvrgnuti hroničnom psihološkom stresu 7 dana uzastopno u trajanjuod 2h. Deplecija makrofaga je vršena intraperitonealnom aplikacijom lipozoma koji sadržeklodronat dva dana pre početka eksperimenta radi uklanjanja rezidentnih makrofaga, a zatim svakitreći dan tokom eksperimenta, u cilju uklanjanja novostvorenih makrofaga. Ćelijske mono- i kokulturesu tretirane eritropoetinom (EPO), kortikosteronom, blokatorom azot-monoksida sintetaze(engl. nitric oxide synthases, NOS) i/ili blokatorom enzimske aktivnosti molekula CD73. Deplecijamakrofaga u koštanoj srži i slezini tretiranih životinja potvrđena je analizom proteina F4/80,primenom Western blota. Za detekciju progenitorskih ćelija eritrocitne loze hematopoezne ćelije sukultivisane na podlozi od metilceluloze obogaćenoj odgovarajućim citokinima. Metodama protočnecitofluorometrije izvršena je detekcija i kvantifikacija eritroblasta u ispitivanim uzorcima koštanesrži i slezine. Specifična aktivnost enzima određena je kolorimetrijski (detekcijom neorganskogfosfata kao proizvod enzimske reakcije). Grisova reakcija je korišćena za određivanje ukupnihnitrata i nitrita (NOx), a koncentracija vanćelijskog adenozin-trifosfata (ATP) je određenaluminometrijski, primenom komercijalnog kita. Koncetracija adenozina je određena primenomtečne hromatografije visoke performanse i masene spektrometrije (engl. ultra high-performanceliquid chromatography-Mass spectrometry, UHPLC-DAD MS/MS). Relativna ekspresija ispitivanihgena je određena metodom Real-Тime PCR. Rezultati su pokazali da je hronični psihološki stressmanjio broj eritrocita i nivo hemoglobina u krvi, a deplecija makrofaga je značajno pogoršalaanemiju kod stresiranih životinja. Hronični psihološki stres je doveo do znatnog povećanja brojaCFU-E i BFU-E ćelija u koštanoj srži i slezini, a deplecija makrofaga je suprimirala ovaj efekatstresa. Deplecija makrofaga u koštanoj srži je prevenirala efekat stresa na CD71+/Ter119+,CD71−/Ter119+ u koštanoj srži i na CD71+/Ter119+ u slezini. Takođe, u uslovima stresa, deplecijamakrofaga je sprečila stresom-izazvano povećanje broja eritroidnih prekursora u okviru E1 i E2subpopulacije u koštanoj srži, kao i povećanje broja E1, E2 i E3 ćelija u slezini... In conditions of disturbed homeostasis, such as bleeding, stress, inflammation, etc., tissuehypoxia occurs, leading to the activation of various mechanisms to maintain adequate oxygensupply. Increased demand for erythrocytes in these conditions results in the activation of stresserythropoiesis (SE). During the SE process, erythroid progenitors migrate from the bone marrow tothe spleen, where these cells affected with bone morphogenetic protein 4 (BMP4) gaincharacteristics specific to stress erythroid progenitors and extend rapidly. In the bone marrow andspleen, erythropoiesis takes place within specialized erythroblastic islands, with macrophagesplaying a crucial role in the formation of mature erythrocytes. In conditions of increased erythrocytedemand, monocytes are recruited to the spleen under the influence of chemokines released from thered pulp, where they differentiate into macrophages within the erythroblastic niche. Besidesmacrophages, a significant component of the hematopoietic microenvironment is composed ofmesenchymal stem cells (MSC), which produce numerous cytokines involved inimmunomodulation, proliferation, differentiation, and migration of hematopoietic cells. Dependingon the microenvironment, MSCs and macrophages establish interactions through various signalingmolecules. However, the role of macrophages and the mechanisms of their interactions with MSCswithin the tissue-specific microenvironment during SE have not been clarified yet. The aim of thisstudy was to investigate the role of macrophages and their tissue-specific interactions with MSCs inthe regulation of stress erythropoiesis, with a particular focus on the involvement of nitric oxide(NO), extracellular nucleotides, and the purinergic signaling system in the examined interactions.For this purpose, male Balb/c mice were used, as well as mono- and co-cultures of RAW264.7 cellsand MSC isolated from the bone marrow and spleen of mice. The mice were subjected to chronicpsychological stress for 7 consecutive days, 2 hours per day. Macrophage depletion was performedby intraperitoneal administration of liposomes containing clodronate two days before the start of theexperiment to remove resident macrophages, followed by administration every third day of theexperiment to eliminate newly formed macrophages. Cell mono- and co-cultures were treated witherythropoietin (EPO), corticosterone, a nitric oxide synthase (NOS) inhibitor, and/or a CD73inhibitor. Macrophage depletion in the bone marrow and spleen of treated animals was confirmedby analyzing the F4/80 protein using Western blotting. To detect erythroid progenitor cells,hematopoietic cells from the erythroid lineage were cultured on methylcellulose-enriched substrateswith appropriate cytokines. Flow cytometry methods were used for the detection and quantificationof erythroblasts in the examined samples of bone marrow and spleen. Enzyme-specific activity wasdetermined colorimetrically by detecting inorganic phosphate as the product of the enzymaticreaction. Gris reaction was used to determine total nitrates and nitrites (NOx), while theconcentration of extracellular adenosine triphosphate (ATP) was determined luminometrically usinga commercial kit. Adenosine concentration was determined using high-performance liquidchromatography and Mass spectrometry (UHPLC-DAD MS/MS). Relative expression of theexamined genes was determined by Real-Time PCR. The results showed that chronic psychologicalstress reduced the number of red blood cells and hemoglobin levels in the blood, and macrophagedepletion significantly alered anemia in stressed animals. Chronic psychological stress led to asignificant increase in the number of colony-forming units-erythroid (CFU-E) and burst-formingunits-erythroid (BFU-E) cells in the bone marrow and spleen, and macrophage depletion suppressedthis stress effect. Macrophage depletion in the bone marrow prevented the stress effect onCD71+/Ter119+ and CD71+/Ter119+ populations in the bone marrow and spleen, respectively.Also, under stress conditions, macrophage depletion prevented stress-induced increases in thenumber of immature erythroid precursors within the E1 and E2 subpopulations in the bone marrow,as well as the increased number of E1, E2, and E3 cells in the spleen... Medicina - Molekularna medicina / Medicine- Molecular medicine Datum odbrane: 04.07.2024. stres eritrocitopoeza, makrofagi, mezenhimalne stromalne ćelije, azot monoksid, purinergički signalni sistem stress erythropoiesis, macrophages, mesenchymal stem cells, nitric oxide, purinergic signaling system 612.119:577.2(043.3) 91552100 193721609 91552101 10357 2026-05-15T10:53:13.053Z 45 no 46 Sanja, 1988- Momčilović 137402889 2024 63 mentor Andrija, 1964- Bogdanović 12651623 2024 63 komentor Sanja, 1976- Vignjević Petrinović 62319113 2024 63 član komisije Marija, 1976- Plješa-Ercegovac 12913511 2024 63 član komisije Danijela, 1977- Leković 52041225 2024 63 član komisije Marija, 1977- Vukelić-Nikolić 27243111 2024 91 str. 5939615 http://phaidrabg.bg.ac.rs/o:38969 no yes 12 2026-05-15T10:53:13.320Z 70 11 1066950 1066998 stres eritrocitopoeza, makrofagi, mezenhimalne stromalne ćelije, azotmonoksid, purinergički signalni sistem stress erythropoiesis, macrophages, mesenchymal stem cells, nitric oxide,purinergic signaling system 612.119:577.2(043.3) 1738 11A32 2024